G-quadruplexes (G4s)

G-quadruplex

• G-quadruplexes (G4s) are stable structures influenced by cations.
• Also called guanosine tetraplexes or G tetraplexes, the orientation of strands in the tetraplex can vary.
• G4s are commonly found in actively transcribed, nucleosome-depleted chromatin regions.
• They are predicted to occur at telomeres, gene promoters, and DNA replication origins.
• RNA G4 enrichment is noted in the 5' and 3' untranslated regions (UTRs) of mRNA.
• RNA G4s (rG4s) play roles in transcriptional regulation, mRNA processing, translation regulation, and RNA translocation.
• rG4s participate in phase transitions and separations within complex macromolecular structures like RNA granules.
• rG4s form scaffolds and may promote neuropathological protein aggregation.
• A potential G4 motif is Gx-N1-7 -Gx-N1-7 -GxN1-7 -Gx, where 'x' ranges from 3-6 and 'N' represents any nucleotide (A, G, T, C, or U).
• G-quadruplex forms:
• Unimolecular G4 (Parallel): Commonly found in mRNA and ncRNA, regulates gene expression.
• Bimolecular G4 (Parallel):Less common and involved in transcription regulation via R-loop G4 formation, and DNA:RNA hybrid G4.
• Tetramolecular G4 (Parallel): Less common tiRNAala, and aids Regulation of stress response.

Ribozymes

• Ribozymes, or ribonucleic acid enzymes, are RNAs that catalyze specific biochemical reactions.
• Catalytic properties stem from RNA's ability to fold into unique secondary structures.
• Ribonuclease P (RNase P) is a ribonucleoprotein particle that catalyzes maturation of the 5' end of tRNA by cleaving precursor sequences.
• RNase P is present in cells across Bacteria, Eukarya, and Archaea domains.

Pseudoknots

• Pseudoknots are RNA structures associated with diverse functions, including non-standard translation mechanisms in viruses like SARS-CoV.

Chromatin Structure

• The human haploid genome contains ~3 billion base pairs of DNA organized into 23 chromosomes.
• Most cells in the body are diploid, containing 23 chromosome pairs, totaling about 6 billion base pairs of DNA per cell.
• Each base pair is about 0.34 nanometers long.
• Total DNA length in a human haploid genome is approximately 1.02 meters.
• During interphase, chromatin is dispersed throughout the nucleus, either as 30nm or 10nm fibers.
• Chromatin states are dynamic, switching between 10nm and 30nm fibers regularly.
• DNA within the 30nm fiber is largely inaccessible while the DNA in the 10nm fiber is more accessible.
• Euchromatin is open chromatin /10nm fiber.
• Heterochromatin is closed chromatin /30 nm fibre.
• Chromatin compaction folds the DNA by around 40X.
• Further organization of chromatin includes loops of 40-90Kb, a protein scaffold, compacts the DNA a further 1000x
• Topoisomerases control DNA coiling and relaxing are important proteins in the scaffold.
• During mitosis and meiosis, DNA compacts substantially (10,000-20,000X) to form chromosomes.
• Chromatin's simplest level of structure is a DNA double helix 2nm wide.
• DNA complexes with histones forms nucleosomes.
• Each nucleosome consists of eight histone proteins wrapped by DNA approximately 1.65 times.
• Nucleosomes fold to form a 30nm fiber.
• Nucleosome core of eight histone molecules along with Histone 1, forms a chromatosome, 11 nm wide.
• The 300-nm fibers compress and are folded to produce a 250-nm-wide fiber, and forms loops averaging approx 300 nm in length.
• The tight coiling of the 250-nm fiber produces the chromatid, a chromosome-like structure that measures approximately 1400 nm in width.

Topoisomerases

• Topoisomerases ensure the manage the topology of folded and intertwined DNA and RNA polymers- They are connected to scaffolding structures and undergo dynamic processing
• TOP2A is found as part of the chromosome scaffold
• TOP1 is located in loop domains, which relievessupercoiling tension.

Nucleosome

• Is the core unit of chromatin.
• A nucleosome has:
• H2A-H2B
• (H3-H4)2,
• H3, H4, H2A, H2B, and DNA.

Histones

• Histones are basic proteins abundant in lysine and arginine, and are found in eukaryotic cell nuclei and most Archaeal phyla.
• Bacteria do not have histones.
• Histones participate in gene expression regulation and DNA replication.
• The five histone families include the following:
  • H1/H5 (linker histones)
  • H2, H3, and H4 (core histones).
• DNA wraps tightly around histones due to electrostatic attraction
• Histones are classified into canonical replication-dependent histones of the cell cycle, and replication-independent histone variants.
• Complex organisms tend to have more variants.
• The H3 histone fold interacts with the H4 fold in a crescent like structure while the H2A and H2B histone folds create are similar.

Histone H1

• The linker histone family, exemplified by histone H1 is closely-related.
• H1's roles are different from those of the core histones.
• H1 linker histones can be removed without disrupting nucleosome structure.
• Nucleosomes with linker histones are called chromatosomes.

Canonical Histone Expression

• Control of histone gene expression occurs at the transcriptional and post transcriptional level
• Histone genes occur in clusters.
• Histone genes do not have introns.
• Histone mRNAs do not have polyA tails.
• SLBP is required for translation of the canonical histone mRNAs

Processing of canonical histone mRNAs

• The conserved stem-loop sequence binds stem-loop binding protein (SLBP).
• The histone downstream element (HDE) base-pairs with U7 small nuclear RNA (snRNA).
• The U7 snRNA is a component of the U7's small nuclear ribonucleoprotein (snRNP).
• CPSF73, along with various factors, is recruited to cleave the pre-mRNA

Histone Variants

• H2AX variant aids DNA repair and DNA recombination.
• H2AZ variant aids gene expression and chromosome segregation.
• Macro H2AA variant aids deacetylation and acetylation status and inactivation of the X chromosome and repression of transcription
• H3.3i variant contributes to transcription activation.
• CENPA variant is associated with assembly of the kinetochore

Histone Post-Translational Modifications(PTMs)

• Lysine PTMs include Acetylation, Acylations, Butyrylation, and Ubiquitin-like modifications etc.
• Non-lysine PTMs include Serotonylation, Dopaminylation, O-palmitoylation etc.
• Key histone tail modification effects include:
• Activation of gene transcription,
• Repression (silencing) of transcription
• Condensation of chromatin into chromosomes (during mitosis and meiosis)
• Facilitation of DNA repair,
• Induction of cell death
• Transcription elongation.
• Histone phosphorylation confers a negative charge resulting in open chromatin
• Is involved in DNA damage repair and chromatin remodelling.